Cancer arises when a normal cell undergoes neoplastic transformation and becomes a malignant cell. Transformed (malignant) cells escape normal physiologic controls specifying cell phenotype and restraining cell proliferation. Transformed cells in an individual's body thus proliferate in the absence of these normal controls, thus forming a tumor.
When a tumor is found, the clinical objective is to destroy malignant cells selectively while mitigating any harm caused to normal cells in the individual undergoing treatment. Chemotherapy is based on the use of drugs that are selectively toxic (cytotoxic) to cancer cells. Several general classes of chemotherapeutic drugs have been developed, including drugs that interfere with nucleic acid synthesis, protein synthesis, and other vital metabolic processes.
5-Fluorouracil (5-FU) is a very widely used drug for the treatment of many different types of cancers, including major cancers such as those of the GI tract and breast (Moertel, C. G. New Engl. J. Med., 330:1136-1142, 1994). For more than 40 years the standard first-line treatment for colorectal cancer was the use of 5-FU alone, but it was supplanted as “standard of care” by the combination of 5-FU and CPT-11 (Saltz et al., Irinotecan Study Group. New England Journal of Medicine. 343:905-14, 2000). Recently, the combination of 5-FU and oxaliplatin has produced high response rates in colorectal cancers (Raymond et al, Semin. Oncol., 25:4-12, 1998). Thus, it is likely that 5-FU will be used in cancer treatment for many years because it remains the central component of current chemotherapeutic regimens. In addition, single agent 5-FU therapy continues to be used for patients in whom combination therapy with CPT-11 or oxaliplatin is likely to be excessively toxic.
5-FU is typical of most anti-cancer drugs in that only a minority of patients experience a favorable response to the therapy. Large randomized clinical trials have shown the overall response rates of tumors to 5-FU as a single agent for patients with metastatic colorectal cancer to be in the 15-20% range (Moertel, C. G. New Engl. J. Med., 330:1136-1142, 1994). In combination with other chemotherapeutics mentioned above, tumor response rates to 5-FU-based regimens have been increased to almost 40%. Nevertheless, the majority of treated patients derive no tangible benefit from having received 5-FU based chemotherapy, and are subjected to significant risk, discomfort, and expense. Since there has been no reliable means of anticipating the responsiveness of an individual's tumor prior to treatment, the standard clinical practice has been to subject all patients to 5-FU-based treatments, fully recognizing that the majority will suffer an unsatisfactory outcome.
The mechanism of action and the metabolic pathway of 5-FU have been intensively studied over the years to identify the most important biochemical determinants of the drug's anti-tumor activity. The ultimate goal was to improve the clinical efficacy of 5-FU by a) modulation of its intracellular metabolism and biochemistry and b) measuring response determinants in patients' tumors prior to therapy to predict which patients are most likely to respond (or not to respond) to the drug. Two major determinants emerged from these studies: 1) the identity of the target enzyme of 5-FU, thymidylate synthase (TS) and 2) the identity of the 5-FU catabolic enzyme, dihydropyrimidine dehydrogenase (DPD).
The first studies in the area of tumor response prediction to 5-FU based therapy centered on the target enzyme TS in colorectal cancer. Leichman et al (Leichman et al., J. Clin Oncol., 15:3223-3229, 1997) carried out a prospective clinical trial to correlate tumor response to 5-FU with TS gene expression as determined by RT-PCR in pre-treatment biopsies from colorectal cancers. This study showed: 1) a large 50-fold range of TS gene expression levels among these tumors, and 2) strikingly different levels of TS gene expression between responding and non-responding tumors. The range of TS levels of the responding groups (0.5-4.1×10−3, relative to an internal control) was narrower than that of the non-responding groups (1.6-23.0×10−3, relative to an internal control). The investigators determined a resulting “non-response cutoff” threshold level of TS expression above which there were only non-responders. Thus, patients with TS expression above this “non-response cutoff” threshold could be positively identified as non-responders prior to therapy. The “no response” classification included all therapeutic responses with <50% tumor shrinkage, progressing growth resulting in a >25% tumor increase and non-progressing tumors with either <50% shrinkage, no change or <25% increase. These tumors had the highest TS levels. Thus, high TS expression identifies particularly resistant tumors. TS expression levels above a certain threshold identified a subset of tumors not responding to 5-FU, whereas TS expression levels below this number predicted an appreciably higher response rate yet did not specifically identify responding tumors.
Subsequent studies investigated the usefulness of DPD expression levels as a tumor response determinant to 5-FU treatment in conjunction with TS expression levels. DPD is a catabolic enzyme which reduces the 5,6 double bond of 5-FU, rendering it inactive as a cytotoxic agent. Previous studies have shown that DPD levels in normal tissues could influence the bio-availability of 5-FU, thereby modulating its pharmacokinetics and anti-tumor activity (Harris et al, Cancer Res., 50: 197-201, 1990). Additionally, evidence has been presented that DPD levels in tumors are associated with sensitivity to 5-FU (Etienne et al, J. Clin. Oncol., 13: 1663-1670, 1995; Beck et al., Eur. J. Cancer, 30: 1517-1522, 1994). Salonga et al, (Clin Cancer Res., 6:1322-1327, 2000, hereby incorporated by reference in its entirety) investigated gene expression of DPD as a tumor response determinant for 5-FU/leucovorin treatment in a set of tumors in which TS expression had already been determined. As with TS, the range of DPD expression among the responding tumors was relatively narrow (0.6-2.5×10−3, 4.2-fold; relative to an internal control) compared with that of the non-responding tumors (0.2-16×10−3, 80-fold; relative to an internal control). There were no responding tumors with a DPD expression greater than a threshold level of about 2.5×10−3. Furthermore, DPD and TS expression levels showed no correlation with one another, indicating that they are independently regulated genes. Among the group of tumors having both TS and DPD expression levels below their respective “non-response cut-off” threshold levels, 92% responded to 5-FU/LV. Thus, responding tumors could be identified on the basis of low expression levels of DPD and TS.
DPD is also an important marker for 5-FU toxicity. It was observed that patients with very low DPD levels (such as in DPD Deficiency Syndrome; i.e. thymine uraciluria) undergoing 5-FU based therapy suffered from life-threatening toxicity (Lyss et al., Cancer Invest., 11: 2390240, 1993). Indeed, the importance of DPD levels in 5-FU therapy was dramatically illustrated by the occurrence of 19 deaths in Japan from an unfavorable drug interaction between 5-FU and an anti-viral compound, Sorivudine (Diasio et al., Br. J. Clin. Pharmacol. 46, 1-4, 1998). It was subsequently discovered that a metabolite of Sorivudine is a potent inhibitor of DPD. This treatment resulted in DPD Deficiency Syndrome-like depressed levels of DPD which increased the toxicity of 5-FU to the patients (Diasio et al., Br. J. Clin. Pharmacol. 46, 1-4, 1998).
Thus, because of a) the widespread use of 5-FU protocols in cancer treatment, b) the important role of DPD expression in predicting tumor response to 5-FU and c) the sensitivity of individuals with DPD-Deficiency Syndrome to common 5-FU based treatments, it is clear that accurate determination of DPD expression levels prior to chemotherapy will provide an important benefit to cancer patients.
Measuring DPD enzyme activity requires a significant amount of fresh tissue that contains active enzyme. Unfortunately, most pre-treatment tumor biopsies are available only as fixed paraffin embedded (FPE) tissues, particularly formalin-fixed parafin embedded tissues which do not contain active enzyme. Moreover, biopsies generally contain only a very small amount of heterogeneous tissue.
RT-PCR primer and probe sequences are available to analyze DPD expression in frozen tissue or fresh tissue. However, those primers are unsuitable for the quantification of DPD mRNA from fixed tissue by RT-PCR. Heretofore, existing primers give no or erratic results. This is thought to be due to the a) inherently low levels of DPD RNA; b) very small amount of tissue embedded in the paraffin; and c) degradation of RNA in the paraffin into short pieces of <100 bp. As a result, other investigators have made a concerted, yet unsuccessful efforts to obtain oligonucleotide primer sets allowing for such a quantification of DPD expression in paraffinized tissue. Thus, there is a need for method of quantifying DPD mRNA from fixed tissue in order to provide an early prognosis for proposed cancer therapies. Because it has been shown that DPD enzyme activity and corresponding mRNA expression levels are well correlated (Ishikawa et al., Clin. Cancer Res., 5:883-889, 1999; Johnson et al, Analyt. Biochem. 278: 175-184, 2000), measuring DPD mRNA expression in FPE specimens provides a way to assess the DPD expression levels status of patients without having to determine enzyme activity in fresh tissues. Furthermore, FPE specimens are readily amenable to microdissection, so that DPD gene expression can be determined in tumor tissue uncontaminated with stromal tissue.
Accordingly, it is the object of the invention to provide a method for assessing DPD levels in tissues and prognosticate the probable resistance of a patient's tumor to treatment with 5-FU based therapies, by determining the amount of DPD mRNA in a patient's tumor cells and comparing it to a predetermined threshold expression level.